PROJECT SUMMARY/ABSTRACT The TGF? signaling pathway has been extensively studied in vascular disease, but there remains considerable controversy regarding the direction and mechanism of effect for how this pathway impacts human coronary artery disease (CAD). Recent genome-wide association studies have identified several loci that harbor TGF? family genes, including the SMAD3 gene at 15q22.33 that encodes a transcription factor critical for converting TGF?-induced cytoplasmic signaling to gene expression changes, and ZEB2 and SKI at 2q22.3 and 1p36.33 respectively, which bind SMAD3 and modulate its transcriptional activity. Studies in this lab have employed histone modification, chromosomal accessibility, allele-specific expression, in vitro genome editing and transgenic reporter mouse studies to identify SMAD3 as the causal gene at 15q22.33. The protective allele for this gene disrupts a potent intronic enhancer in the SMAD3 gene that is associated with decreased SMAD3 expression in vascular tissues, suggesting that expression of SMAD3 in SMC promotes risk for CAD. In SMC, TGF? signaling is known to have important differentiative and anti-proliferative roles during vascular development, but this function may be deleterious in the disease setting where SMC dedifferentiation and proliferation, ?phenotypic modulation,? allows this cell type to bolster structural integrity and retard plaque rupture. A disease-promoting role for TGF? is supported by our studies with TCF21, a CAD associated transcription factor that promotes SMC phenotypic modulation and whose protective allele confers increased expression. Taken together, these data suggest our Central Hypothesis: the TGF? signaling molecule SMAD3, in conjunction with ZEB2 and SKI, regulates a transcriptional network that mediates the adaptive SMC phenotypic response to vascular stress, with allelic variation modulating this response contributing to CAD risk. Experiments proposed in Aim 1 in the ApoE-/- atherosclerosis mouse model will evaluate disease anatomy with SMC-specific deletion of Smad3, along with human risk and protective haplotypes created by genome editing. Lineage tracing and single cell RNA-seq will define the role of Smad3 in regulating the cellular response to disease stimuli, and molecular phenotype as lesion SMC dedifferentiate and contribute to the macrophage lineage. In Aim 2, ChIP-seq and RNA-seq studies in human coronary artery SMC will define the network of genes that are regulated by SMAD3 and investigate how expression of ZEB2 and SKI modulates the molecular composition of this network. In vitro studies in human coronary artery SMC in Aim 3 will identify the cellular and molecular processes that are mediated by SMAD3 and how these functions are modified by ZEB2 and SKI. Taken together, these studies will significantly advance our understanding of how SMAD3 and related factors ZEB2 and SKI govern the SMC phenotypic response to vascular disease, and how perturbation of their function contributes to CAD risk.